Fig 1.
The effect of forskolin on the IL-1β-induced increase in OTR mRNA and protein levels and the impact of PKA knockdown.
Myometrial cells were isolated as described above in Materials and Methods, and treated with IL-1β (1ng/mL) and/or forskolin (100μM) either alone or in combination for 6 hours. mRNA and protein were extracted, and the levels of OTR (A) mRNA were measured using quantitative rt-PCR. The levels of OTR (B) protein were measured using Western blotting. A representative western blot is shown next to the graph displaying the densitometry of the protein levels. PKAC-α was knocked down using siRNA (siPKAC-α) controlled with non-targeted siRNA [siNT]. Representative western blots to demonstrate transfection is shown above. After transfection, cells were incubated for 96 hours before being treated with progesterone (10μM), IL-1β (1ng/mL) and forskolin (100μM) in combination for 6 hours. The mRNA was extracted, and the levels of OTR (C, D), mRNA were measured using rt-PCR. Data are shown as the mean and SEM. A and B were compared using Wilcoxon matched pairs test for data that were not normally distributed and paired t test for data that were normally distributed. C and D were compared using a non-parametric paired test on the basis that we wanted to understand the impact of blocking PKA. *P<0.05, **P<0.01, ***P<0.001 (n = 8–9 myometrial samples from 8–9 different women in each experiment).
Fig 2.
Forskolin represses IL-1β-induced total and nuclear and cytosolic p65 protein and c-jun levels.
Myometrial cells were isolated from myometrial biopsies obtained from women at the time of pre-labor term Caesarean section as described above in Materials and Methods, and treated with IL-1β (1ng/mL) and/or forskolin (100μM) either alone or in combination for 6 hours. Protein was extracted, and the levels of total p65 (A), and total c-jun (B) protein expression were measured using western blotting. Myometrial cells were isolated as described above in Materials and Methods, and treated with forskolin (100μM) or IL-1β (1ng/mL) either alone or in combination for 1 hour. Cells were lysed and samples purified for cytoplasmic or nuclear protein. Western blotting was performed using antibodies directed against phospho-p65 (Ser536) (C, D), and phospho c-jun (E, F). TATA-binding protein (TBP) and α-tubulin were used as the internal controls for nuclear and cytosolic fraction, respectively. MKP-1 (G, H) and IKBα (I, J) mRNA and protein levels were measured using rt-PCR and western blotting respectively. Data are shown as the mean and SEM, and were compared using (control vs. forskolin and IL-1β alone vs. IL-1β + forskolin) Wilcoxon matched pairs test for data that were not normally distributed and paired t test for data that were normally distributed. *P<0.05, **P<0.01, ***P<0.001 when compared to control. (n = 6–7 myometrial samples from 6–7 different women in each experiment).
Fig 3.
The effect of the combination of forskolin and progesterone on the IL-1β-induced increase in OTR mRNA and protein levels.
Myometrial cells were isolated as described in Materials and Methods, and treated with progesterone (10μM), forskolin (100μM) or IL-1β (1ng/mL) either alone or in combination for 6 hours. mRNA and protein were extracted, and the levels of OTR (A) mRNA were measured using quantitative rt-PCR and the levels of OTR (B) protein were measured using Western blotting. A representative western blot is shown next to the graph displaying the densitometry of the protein levels. Data are expressed as mean SEM and were compared using Friedman’s Test, with a Dunn's Multiple Comparisons post hoc test for data that were not normally distributed, and using ANOVA, with Dunnett and Bonferroni’s post-test for data that were normally distributed, *P<0.05, **P<0.01 (n = 8–9 myometrial cells from 8–9 different women).
Fig 4.
The combination of cAMP with progesterone represses IL-1β-induced nuclear transfer of phospho c-jun and phospho p65.
Myometrial cells were isolated from myometrial biopsies obtained from women at the time of pre-labor term Caesarean section as described above in Materials and Methods, and treated with progesterone (10μM), forskolin (100μM) or IL-1β (1ng/mL) either alone or in combination for for 0 minute, 30 minutes, 1 hour, 2 hours and 6 hours. Cells were lysed and samples purified for cytoplasmic or nuclear protein. Western blotting was performed using antibodies directed against phospho c-jun (A, B), and phospho-p65 (Ser536) (C, D). α-tubulin and TATA-bind protein (TBP) were used as the internal controls for cytosolic and nuclear fraction, respectively. Data were compared using Friedman’s Test, with a Dunn's Multiple Comparisons post hoc test for data that were not normally distributed, and using ANOVA, with Dunnett and Bonferroni’s post-test for data that were normally distributed, *P<0.05, **P<0.01, ***P<0.001. (n = 6 myometrial cells from 6 different women and only 30 minutes, 1 hour, and 2 hours time points were shown in the figure above).
Fig 5.
The effect of PKA knockdown on the repression of the IL-1β-induced increase in OTR mRNA and protein levels by forskolin and progesterone.
Myometrial cells were isolated from myometrial biopsies obtained from women at the time of pre-labor term Caesarean section as described above in Materials and Methods. After the cells were about 80% confluent, PKAC-α were knocked down using siRNA (siPKAC-α, efficacy shown in inset figure) controlled with non-targeted siRNA [siNT]. Cells were knocked down using siNT. Representative western blots to demonstrate transfection is shown above. After transfection, cells were incubated for 96 hours before being treated with progesterone (10μM), IL-1β (1ng/mL) and forskolin (100μM) in combination for 6 hours. The mRNA was extracted, and the levels of OTR (A, B) mRNA were measured using rt-PCR. Data were compared using Friedman’s Test, with a Dunn's Multiple Comparisons post hoc test for data that were not normally distributed, and using ANOVA, with Dunnett and Bonferroni’s post-test for data that were normally distributed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n = 6–7 myometrial cells from 6–7 different women).